Most of the accomplishments of this project have now been presented in scientific periodicals. Two new areas of investigation of synaptic structure are underway. New methods have been developed to use rapid freezing to determine how the distribution of calcium in synapses changes in different functional states. Organelles which store and release calcium during secretion as well as sequestering it afterwards have been identified. This work is significant in that it defines the dynamic structure of normal synapses by relating normal variations in structure to different functional states. These approaches to determining the distribution of soluble substances are also being applied to use a new antimony-labeled acetylcholine molecule to determine where acetylcholine is taken up and stored in cholinergic synaptosomes from squid brain. A new method of staining freeze substituted tissue has recently been developed which requires no further stain after the sections are cut, so the stain extends evenly through the section. Therefore the three dimensional structure of the cytoskeleton and related fine filaments in synapses can be determined in continuous serial sections. The locations in the synapses of these filaments by calciuim activated proteases is thought to be an important determinant of synaptic shape and function. Differences in the organization of the cytoskeleton and related filaments during synaptic activity are now under investigation in order to determine their role in mobilizing synaptic vesicles for release.